Toxicological profile of the aqueous-fermented extract of Musa paradisiaca in rats.

Objective
This study was conducted to assess the toxicity profile of the aqueous-fermented extract of Musa paradisiaca in rats.


Materials and Methods
In acute toxicity test, the rats of different groups were orally administered with a single dose of 500, 1000, 2000 and 5000 mg/kg of fermented extract of M. paradisiaca. The rats were monitored for behavioral changes, toxicity signs and mortality. In sub-acute test, the rats were orally administered with fermented M. paradisiaca extract (200, 400 and 800 mg/kg/day) for 14 days. Haematological and serum biochemical parameters were evaluated and histopathological studies of the liver and kidney were done. The study was performed from June to July 2017.


Results
Concerning the acute toxicity, no toxicity signs or death were recorded and an LD50 value of >5 g/kg for fermented extract of M. paradisiaca was observed. Regarding the sub-acute toxicity, ingestion of the fermented extract of M. paradisiaca caused no significant effects (p<0.05) in terms of relative organ weight, body weight percentage, haemoglobin, red blood cells count, electrolytes levels, lymphocytes count, basophils count, and aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels. However, significant differences (p<0.05) were observed in white blood cells, eosinophils, platelets, neutrophils and monocytes counts, and urea, creatinine, alanine aminotransferase (ALT) and high-density lipoprotein (HDL) levels. The histological assessments of the liver and kidney showed normal results.


Conclusion
The findings of this study has suggested that daily administration of fermented extract of M. paradisiaca at doses up to 800 mg/kg for 14 days, is not toxic and may be considered safe for therapeutic uses.

Traditional medicine (TM) is comprised of explicable and inexplicable knowledge, acquired skills, and sometimes beliefs from different cultures of the world which are used in the treatment of diseases and maintenance of good health. TM is also designated as complementary and alternative medicine (CAM) and/or nonconventional medicine in developed countries (Zhang, 2000). In Africa, more than 80 % of the population use TM while in developed countries such as Canada, up to 70% of the population use CAM for their healthcare needs (World Health Organization, 2002) because of its accessibility, affordability, efficacy, bioavailability and less side effects (Elujoba et al., 2005;Ezekwesili et al., 2014;Elujoba et al., 2005;Ugbogu et al., 2016).
Due to the presence of the several nutrients including minerals and bioactive metabolites in M. paradisiaca, virtually all parts of the plant have been used for management of various ailments. For example, aqueous extract of fermented unripe M. paradisiaca fruits and unripe M. paradisiaca peels possess anti-ulcerogenic (Ezekwesili et al., 2014;Ikpeazu et al., 2017) and anti-microbial (Kapadia et al., 2015) activities. Studies have also shown that aqueous extract of M. paradisiaca fruit pulp possesses antioxidant (Shodehinde and Oboh, 2013) and wound healing properties (Agarwal et al., 2009) and could be used as a remedy against diabetes and hepatic dysfunction (Ojewole and Adewunmi, 2003;Eleazu and Okafor, 2015). Extracts of M. paradisiaca stem are used for wound healing and have been shown to have antimicrobial activity (Amutha and Selvakumari, 2016) and hepato-protective property (Nirmala et al., 2012). M. paradisiaca flower possesses antimicrobial and anti-hyperglycemic (Jawla et al., 2012) as well as antidiarrhoea effects (Yakubu et al., 2015). Amongst all these potential uses of M. paradisiaca for management and treatment of diseases, there is little or no published data on comprehensive assessment of the toxicity of aqueous extract of fermented M. paradisiaca which is currently used in treating ulcers. A previous investigation (Ikpeazu et al., 2017) revealed that the effectiveness of fermented products over the non-fermented counterparts is due to the synergistic role of phytoconstituents and microbiomes.
Probiotics, as live microorganisms, have been observed to confer health benefits such as enhancement of mineral metabolism, reduction of LDL-C level, stimulation of vitamin B-complex biosynthesis, modulation of the immune system, anti-hypertensive and antiulcerogenic effects (Musa et al., 2009;Liong et al., 2009;Haukioja, 2010). It is against this backdrop that the current study was designed to assess the toxicity profile of aqueous-fermented extract of M. paradisiaca concerning body weight, relative organ weight, and histopathological indices as well as haematological, hepatocellular, and lipid profiles in rats.

Sample collection and identifications
Fresh unripe plantain (M. paradisiaca) was purchased from Eke Okigwe Market, Okigwe Local Government Area in Imo State. It was authenticated by a Botanist as Musa paradisiaca; a sample was kept at the herbarium of University of Nigeria Nsukka with voucher number UNH No.: 812.

Sample preparation
The rotten fruits were removed, and the rest were washed with clean water. M. paradisiaca were peeled, washed and cut into approximately 4-mm long pieces. With the aid of G & G® Electronic scale, 200 g of the sliced M. paradisiaca were weighed in a beaker and 300 ml of distilled water was added. The beaker containing the plantain and water was covered and allowed to stand at room temperature (25 o C), overnight (i.e. from 6 pm to 8 am). After 15 h of fermentation, the extract was filtered using cheesecloth and the filtrate was used immediately.

Animal handling
A total of 74 healthy rats (150-200g) comprising 50 males and 24 female rats were purchased from University of Nigeria Nsukka and transported to Biochemistry Department, Abia State University Uturu, Nigeria. The rats were allowed to acclimatize for two weeks in a wellventilated house with clean cages under normal environmental conditions of temperature (25-28 o C) and humidity (35-60 %) with 12 h/12 h light/dark cycles, before the start of the experiment. The rats were fed with standard commercial food and they had free access to water. Strict adherence to ethical principles (Neuwinger 2000), of the World Health Organization of good laboratory practices and United States guidelines for animal experiments (Care, Animal, and Use Committee,1998, CNRC, 2010 was maintained in this study. Experimental procedures and animal handling were approved by the Abia State University Research Ethical Clearance Committee -ABSU/REC/BMR/015.

Acute toxicity test (median lethal dose (LD 50)
A total of 50 rats were divided into five experimental groups of ten (10) rats and each group consisted of five (5) female and five (5) male rats. The rats were fasted overnight and a single dose of 500, 1000, 2000 or 5000 mg/kg aqueous-fermented extract of M. paradisiaca fruit pulp was orally (i.e. gavage) administered to groups B, C, D and E, respectively while group A received 0.25 ml of distilled water and served as the control. The rats were monitored for behavioral changes, toxicity signs and mortality for 24 h and thereafter for 14 days (OECD, 2001) guideline 423 with little modifications.

Sub-acute toxicity study
Twenty-four (24) male rats were allocated into four (4) experimental groups each consisted of six (6) rats. Aqueousfermented extract of M. paradisiaca fruit pulp (200, 400, or 800 mg/kg) or 0.25 ml of distilled water for the control group, were administered once daily for 14 consecutive days (OECD, 1995) guideline 407.

Organs and blood sample collection
After 14-days administration of aqueous-fermented extract of M. paradisiaca fruit pulp the rats were anesthetized and sacrificed immediately on the 15 th day of the experiment. Blood samples were collected via cardiac puncture. The samples for biochemical tests were dispensed into heparinized containers while the samples for haematological analysis were collected into ethylenediaminetetraacetic acid (EDTA)containers. The heart, kidney, liver, lungs and spleen were carefully removed by dissection and their weights were determined.

Clinical chemistry studies
Liver enzymes including alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP); renal function parameters including bicarbonate (HCO3 -), chloride (Cl -), creatinine, potassium (K + ), sodium (Na + ) and urea and lipid profile parameters including triglyceride, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C), were evaluated. All the abovementioned parameters were evaluated using ready-to-use kits obtained from Randox Laboratory Ltd. Co. Antrim, United Kingdom based on the manufacturer's instructions.

Histological studies
The liver and kidney were fixed in 10 % formalin after the rats were sacrificed, thereafter, samples were cleaved, processed and embedded in paraffin wax. Then, the tissues were sectioned into 5-m thickness specimen, stained with haematoxylin and eosin and evaluated under an optical microscope by an experienced pathologist as described by Fisher et al. (2002).

Statistical analysis
The results of this study were presented as mean ± standard deviation of three replicates using excel package. The experimental data were tested for homogeneity of variance and then subjected to one-way analysis of variance (ANOVA) and the difference between the samples mean were tested by Tukey post-hoc test using R-statistics software version 3.03. A p≤0.05 was considered statistically significant.

Results
In acute toxicity study, any signs of abnormalities before and after treatment with aqueous-fermented extract of M. paradisiaca fruit pulp (500-5000 mg/kg) were recorded. No death or signs of toxicity were observed in all the groups. However, rats fed with 2000 mg/kg extracts were calm within 2 h of administration while rats treated with 5000 mg/kg could not eat enough food (Table 1). In sub-acute toxicity test, no significant differences (p<0.05) were observed in percentage of weight gain and relative organ weight of all the groups (Table 2 and Figure 1). No significant differences (p<0.05) were observed in the blood profile in terms of Hb, PCV, MCH and MCV while significant differences (p<0.05) were observed in WBC and platelet counts of rats fed with 200, 400, 800 mg/kg of aqueous-fermented extract of M. paradisiaca compared to the control group (Table 3).  Values represent mean ± SD of n=6 rats in each group. Values represent mean ± SD of n=6 rats in each group. Significant differences compared to control at p<0.05 are indicated with *, **and *** showing the level of significance.
Significant decreases (p<0.05) in urea and creatinine levels were observed in the rats fed with aqueous-fermented extract of M. paradisiaca at all concentrations while Clsignificantly reduced only at the dose of 800 mg/kg. No significant changes were observed in the serum levels of K + , Na + and HCO3compared to the control group ( Figure 2). ALT showed a significant decrease in rats treated with aqueous-fermented extract of M. paradisiaca at all concentrations while ALP value ranged from 89.80±0.17 to 102.50±0.40 U/L and AST ranged from 9.67±2.08 to11.00±1.00 U/L (Figure 3). Values represent mean ± SD of n=6 rats in each group. Significant differences compared to control at p<0.05 are indicated with asterisk *.
Lipid profile investigation showed that cholesterol, LDL-C and HDL-C increased significantly (p<0.05) in groups treated with 400 and 800 mg/kg extract of M. paradisiaca compared to the control group ( Figure 4). There were no observable changes in histology of the rats' liver and kidney (Figures 5 and 6).

Discussion
In folk medicine, various parts of M. paradisiaca are used in the management or treatment of many ailments such as diabetes, diarrhea, burns, hypertension, marasmus, bites, hemorrhage and ulcers. Ikpeazu et al. (2017) established that aqueous-fermented extract of M. paradisiaca has an effective anti-ulcer potential. However, regardless of various medicinal uses of M. paradisiaca, there is little or no comprehensive report on the toxicity profile of aqueous-fermented extract of M. paradisiaca fruit pulp. Aqueous-fermented extract of M. paradisiaca fruit pulp is used in Southeastern parts of Nigeria to treat ulcers. However, administration of this extract over a long period of time may be detrimental in the absence of proper dose monitoring or without considering its toxic effects. This study therefore investigated acute and sub-acute toxicity profile of aqueous-fermented extract of M. paradisiaca in rats.
Toxicity studies of substances involve acute, sub-acute, chronic, or sub-chronic toxicity (Balogun and Tom Ashafa, 2016; Kong et al., 2016b). In acute toxicity studies, oral administration of fermented M. paradisiaca 500 and 5000 mg/kg to rats did not cause mortality nor toxicity signs. However, behavioral changes were observed at the doses of 2000 mg/kg (rats were calm within 2 h) and 5000 mg/kg (rats lost appetite for food). This is an indication that the LD50 of M. paradisiaca extract is well above 5000 mg/kg and an LD50 of >5000 mg/kg has been reported to be safe and can be considered non-toxic (OECD, 2001). Determination of organ weight in in vivo toxicity studies is essential for assessment of animals' sensitivity to toxicity, physiologic perturbations, induction of enzymes, and acute organ damage (Michael et al., 2007). In sub-acute toxicity studies, non-significant differences (p<0.05) in weight gain (Table 2) and relative organ weight (Figure 1) were observed in all groups demonstrating normal weight increase in all groups.
Blood is regarded as an essential biological sample for assessment of physiological, nutritional and pathological status of vertebrates. Investigation of blood parameters are also useful for determination of the impact of potentially toxic substances on blood parameters (Han et al., 2010). Significant increases (p<0.05) which were within the internationally accepted range of the proportions of neutrophils, eosinophil and monocytes and non-significant changes in lymphocytes and basophils counts suggest that this extract did not cause any damage to the tissues nor the immune system. Significant increases in platelet counts observed in all extract-treated groups compared to control group, are possibly because of elevated secretion and production of thrombopoietin, a hormone responsible for the synthesis of platelets (Kaushansky, 1995). Hepatic and renal functions impairment caused by toxicants could be revealed by investigation of blood and biochemical parameters as liver and kidney are pivotal for survival (Olorunnisola et al., 2012). ALP, AST and ALT are essential biomarkers of cellular integrity and function of the liver and heart, which are often released into the blood from damaged liver (Chavda et al., 2010). Cellular damage, tissue necrosis and cardiovascular diseases lead to elevation of serum concentrations of ALT and AST (Ioannou et al., 2006;Adeyemi et al., 2015). In this study, serum levels of ALT significantly reduced (p<0.05) while AST slightly decreased, suggesting that aqueousfermented extract of M. paradisiaca may possess some hepato-protective properties.
Increases or decreases in serum electrolytes level may be caused by a hypoor hyper-functioning organ or tissue. Kidney functions are commonly investigated by assessing the level of sodium, potassium, and chlorides in blood serum (Balogun and Tom Ashafa, 2016). The serum concentration of urea and creatinine decreased significantly (p<0.05) while HCO -3, K + and Na + levels were not significantly different from those of the control group (p<0.05), indicating that M. paradisiaca has no adverse effect on the kidney. Although, significant increases (p<0.05) were observed in total cholesterol, HDL-C and LDL-C levels, the observed increases in values were within the normal range. The histological assessment of the liver and kidney showed normal architecture of the organs without any detrimental pathological changes confirming non-toxic nature of M. paradisiaca at tested doses (Figures 5 and  6).
In conclusion, this study revealed that the LD50 of aqueous-fermented extract of M. paradisiaca is above 5g/kg. The study also established that the oral administration of aqueous-fermented extract of M. paradisiaca up to the dose of 800 mg/kg for 14 days, is not toxic and therefore may be considered safe for therapeutic uses. This study also revealed that aqueous-fermented extract of M. paradisiaca decreased liver biomarker enzymes (e.g. ALT, AST and ALP) when compared to their respective controls, suggesting that aqueousfermented extract of M. paradisiaca could have hepato-protective effects.